The product of the adenovirus ElA gene is a nuclear oncoprotein that has several biological properties including transcriptional stimulation, transcriptional repression, and cell transformation and immortalization. This proposal examines two aspects of ElA, the first of which is the mechanism of nuclear import. ElA has two discrete regions that are responsible for nuclear localization, one that directs nuclear import in all cells and a second that directs nuclear import in oocytes and pre-neurula embryonic cells, but not post-neurula cells or adult somatic cells. The fine structure of the second region, termed the developmentally regulated nuclear localization signal (drNLS) has been precisely defined and shown to be quite different from other NLSs in that it is devoid of basic residues and is hydrophobic. An hypothesis to explain the function of the drNLS is that it interacts with a unique developmentally regulated cytoplasmic carrier protein. By a variety of biochemical methods, the carrier protein will be isolated, cloned, and tested for importance during development. Transcriptional stimulation by ElA requires the coordinate activity of several cellular proteins that bind discrete promoter regions. One of these factors, activating transcription factor (ATF), has been partially cloned from Xenopus. Developmental profiles of ATF RNA and protein show these molecules to be very low in oocytes and blastulae, increase dramatically in gastrulae, and drop precipitously thereafter. These data suggest that ATF has an important function during gastrulation. This will be explored further by first analyzing ATF spatial expression during Xenopus development. To determine whether ATF is necessary for normal development, a surrogate genetic approach will be employed. A full-length ATF cDNA clone will be isolated, sequenced, and used to misexpress and overexpress wild type and mutant proteins in embryos. An analysis of the resulting phenotype will indicate the developmental process with which ATF is involved. ElA is a well-known modulator of cell growth control. An analysis of the mechanisms by which ElA comes to reside in the nucleus, its site of action, and an examination of the cellular factors that are influenced by the protein enhances our understanding of cancer.
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